The present invention is generally in the field of methods and compositions for treating metabolic disorders, particularly diabetes and obesity.
Insulin Dependent Diabetes Mellitus (IDDM) is a common condition that results in both high morbidity and high mortality despite the general availability of insulin and the awareness that intensive insulin therapy can maintain euglycemia and significantly reduce complications. The major limitation is that the maintenance of euglycemia requires a regimental approach to monitoring blood glucose, and appropriately administering and tailoring the doses of insulin. Although the use of insulin, has revolutionized the treatment of diabetes, the need for repeated injections, continuous monitoring followed by appropriate dose changes, leads to poor patient compliance and lack of adequate 24 hour control.
Alternative treatments for diabetes include islet cell transplantation, however, this approach has limitations. Specifically, the transplantation requires some invasive surgical intervention and necessitates an immunosuppressant therapy. Moreover, the results from islet cell transplantation have generally been disappointing. Other methods involve ex vivo gene therapy of host cells which may avoid the immune rejection. With ex vivo gene therapy, cells are extracted from an individual and subjected to genetic manipulation by inserting genetic material into the cells. The genetically manipulated cells are implanted back into the individual from which they were removed. The genetic material is then expression in vivo. Although this approach has resulted in successful in vivo expression of the protein, this approach also has limitations, for example, the genetically engineered cells may either be targeted by the underlying autoimmune disease, or undergo uncontrolled cell division and thereby induce tumor growth.
Gene transfer for the treatment of diabetes has been demonstrated using cationic liposome:DNA complexes as described by Leibiger et al. (1991) Biochem. Biophys. Res. Comm. 174: 1223-1231. Other studies have shown systemic gene expression after intravenous injection of an expression plasmid:cationic liposome complex (Zhu et al., (1993) Science 261, 209-211). However, the results demonstrate poor efficiency of transfection, and a reduction in expression over time. Sugiyama et al. generated an adeno-associated virus (AAV) vector expressing rat insulin (Sugiyama et al. (1997) Horm. and Metab. Res. 29, 599-603). These authors demonstrated insulin expression in primary hepatocytes in vitro and then further showed that the vector could be introduced in vivo by direct injection into the liver of diabetic mice. The results of the study showed a mild drop in plasma glucose at 5 days. However, the results demonstrated a transient efficacy reflected by the low titer of vector stocks ( less than 107/ml), as well as instances of hepatic injury following direct intraparenchymal injection, and low transduction efficiency.
Therefore, a need exists for an in vivo gene therapy approach whereby the target cell is transduced and provides long term, regulatable expression of a target protein.
The invention provides methods and compositions for targeting endocrine cells capable of converting an immature expressed protein into a mature protein, and secreting the expressed protein into the systemic circulation. The invention is based, in part, on the discovery that endocrine cells, in particular the cells of the diffuse neuroendocine system (DNES), can be modified to incorporate a nucleic acid molecule which expresses a protein with a desired therapeutic effect on a subject. The invention features targeting endocrine cells capable of converting an immature expressed protein into a mature protein.
In particular, the invention features methods and compositions for treating disorders, such as diabetes and obesity by modifying endocrine cells to incorporate a nucleic acid molecule which expresses a desired protein. The expressed protein can be secreted into the systemic circulation to obtain therapeutic blood levels of the protein thereby treating the subject in need of the protein. The transformed endocrine cells provide long term therapeutic effects for disorders associated with a deficiency or misregulation of a particular protein.
In one aspect of the invention, an orally administered vector expressing insulin can be used to target cells which can subsequently secrete the expressed insulin into the blood circulation. More specifically, targeting secretory cells that are capable of converting immature proteins to mature proteins, such as specific cells of the intestine, e.g., DNES cells. The DNES cells are capable of processing pro-insulin into mature insulin by virtue of these cells expressing enzymes that are capable of converting pro-insulin to insulin, e.g., the enzyme, convertase. The insulin is then released into the portal circulation and is able to lower blood glucose.
The invention provides a direct in vivo gene therapy approach that has several advantages over both transplantation and ex vivo gene transfer techniques. Specifically, in vivo somatic cell gene transfer can be used to directly introduce the human insulin gene into host cells without disrupting normal anatomy and without the need for surgical intervention, or the need for immunosuppression to prevent rejection. A continuous low level of endogenous insulin may contribute to an improvement in glycemic control and can delay the onset or slow the progression of diabetic complications. In addition, the subject with diabetes would be protected from ketoacidosis.
Accordingly, the invention features a method for inducing expression of a protein in a specific tissue comprising:
identifying a tissue that is capable of enzymatically converting an immature protein into a mature protein, and is also capable of secreting the mature protein from the cells into the systemic circulatory system;
orally administering to the tissue a viral vector comprising a tissue specific promoter and a nucleic acid encoding a protein of interest;
expressing the protein of interest in the tissue such that if the expressed protein is an immature protein, the tissue enzymatically converts the immature protein into a mature protein prior to secreting the mature protein from the cells into the systemic circulatory system.
In one embodiment, the specific tissue is an endocrine tissue, for example, the intestinal lining and endocrine tissue comprising the diffuse neuroendocine system (DNES) cells. The protein of interest can be selected based on the disorder that requires treatment. If the disorder is diabetes, the protein of interest is insulin or pro-insulin.
In one embodiment, the viral vector is selected from the group consisting of an adeno-associated vector, a parvovirus vector, an adenovirus vector, a herpes virus vector and a lentivirus vector. In a preferred embodiment, the viral vector is an adeno-associated vector.
In another embodiment, the tissue specific promoter is selected from the group consisting of insulin promoter, glucokinase promoter, L-pyruvate kinase promoter, glucagon promoter, elongation factor 1 alpha promoter, and the rat insulin promoter. In a preferred embodiment, the tissue specific promoter is the elongation factor 1 alpha promoter. In another preferred embodiment, the tissue specific promoter is the rat insulin promoter.
In another aspect, the invention features a method for delivering a protein of interest to the systemic circulatory system of a subject comprising:
orally administering a nucleic acid construct into an endocrine tissue in vivo, wherein the nucleic acid construct comprises a nucleotide sequence encoding a protein of interest operably linked to a promoter specific for the endocrine tissue; and
expressing the protein of interest in the endocrine tissue such that if the expressed protein is an immature protein, the endocrine tissue comprises at least one enzyme capable of enzymatically converting the immature protein into a mature protein, and wherein the endocrine tissue secretes the mature protein into the systemic circulatory system, to thereby deliver a protein of interest into the systemic circulatory system.
In a preferred embodiment, the step of orally administering the nucleic acid construct comprises administering the nucleic acid construct into the intestine.
In another aspect, the invention features a method for treating diabetes comprising:
orally administering a nucleic acid construct into DNES cells in vivo, wherein the nucleic acid construct comprises a nucleotide sequence encoding insulin operably linked to a promoter specific DNES cells;
expressing a therapeutically effective amount of biologically active insulin in the DNES cells; and
secreting the expressed insulin into the systemic circulatory system, to thereby treat diabetes.
In another aspect, the invention features a method for treating diabetes comprising:
orally administering a nucleic acid construct into DNES cells in vivo, wherein the nucleic acid construct comprises a nucleotide sequence encoding pro-insulin operably linked to a promoter specific DNES cells;
expressing a therapeutically effective amount of biologically active pro-insulin in the DNES cells;
converting the pro-insulin to insulin in the DNES cells; and
secreting the insulin into the systemic circulatory system, to thereby treat diabetes.
In one embodiment, the insulin is expressed in the DNES cells for at least 3 months. In another embodiment, the insulin is expressed in the DNES cells for at least 6 months. In another preferred embodiment, the DNES cells convert the pro-insulin to insulin using the convertase enzyme.
In another aspect, the invention features a method for regulating blood glucose levels in a subject comprising:
orally administering a nucleic acid construct into DNES cells in vivo, wherein the nucleic acid construct comprises a nucleotide sequence encoding insulin operably linked to a glucose responsive promoter;
expressing a therapeutically effective amount of biologically active insulin in the DNES cells; and
secreting the expressed insulin into the systemic circulatory system in an amount sufficient to regulate blood glucose levels.
In one embodiment, the glucose responsive promoter is selected from the group consisting of insulin promoter, glucokinase promoter, L-pyruvate kinase promoter, glucagon promoter, and the rat insulin promoter. In another embodiment, the blood glucose levels are reduced to normal blood glucose levels. In yet another embodiment, the insulin is secreted in response to high blood glucose levels.
In another aspect, the invention features a AAV viral vector for expression of a insulin in the DNES cells comprising a tissue specific promoter and a nucleic acid encoding the insulin.
In a preferred embodiment, the promoter is an insulin promoter.